1. Field of the Invention
The invention is related to and has among it objects a novel bacterial strain and use thereof to reduce limonoid bitterness in citrus products and by-products, particularly citrus juices.
2. Description of the Prior Art
Bitterness due to limonoids in certain citrus juices is one of major problems of the citrus industry worldwide and has significant economic impact. At least 29 limonoids have been isolated from citrus and citrus hybrids; four of them, limonin, nomilin, ichangin and nomilinic acid, are bitter. Limonin is the principal bitter limonoid constituent of citrus juice and the primary cause of the bitterness problem. Because of its intense bitterness, only a small amount of limonin is needed to render the juice unpalatable. Recently, nomilin has been shown to also cause the bitterness problem in certain juices.
The intact fruit does not normally contain limonin, but rather a nonbitter precursor, limonoate A-ring lactone. This precursor converts to limonin in the juice soon after extraction. This conversion proceeds under acidic conditions and is also accelerated by the action of limonin D-ring lactone hydrolase which has been shown to be present in citrus. This phenomenon of delayed bitterness is a serious economic problem to the citrus industry and many methods to prevent or remove limonin bitterness have been tried, including treatment of the fruit with ethylene gas, selective adsorption of limonin from juice using cellulose acetate beads, and use of limonin bitterness suppressing agents in the juice. None of these methods has proved entirely satisfactory.
Several species of bacteria capable of metabolizing limonoids have been isolated and treatment of citrus juice with limonoid-metabolizing bacteria or enzymes isolated from such bacteria has been carried out to reduce limonin bitterness. Hasegawa et al., Journal of Agricultural and Food Chemistry, Volume 30, pp 509-511 (1982) describes limonin debittering of naval orange juice using Arthrobacter globiformis cells cultured on a nutrient medium containing sodium limonoate and subsequently immobilized in acrylamide gel. Vaks and Lifshitz, Journal of Agricultural Chemistry, Volume 29, pp 1258-1261 (1981) discloses the use of Acinetobacter sp. cultured on a medium containing limonin to debitter orange juice. Their treatment methods included use of the free cells, immobilization of the cells in acrylamide or agarose and entrapment in a dialysis sac. U.S. Pat. Nos. 3,869,345 and 3,911,103 (Hasegawa et al.) disclose the preparation of the enzyme, limonoate dehydrogenase from Arthrobacter globiformis cultured on a nutrient medium containing limonoate and use of the enzyme to reduce the development of bitterness in citrus products containing the limonin precursor, limonoate A-ring lactone. U.S. Pat. Nos. 3,917,512 and 3,920,851 (Hasegawa et al.) disclose preparation of the enzyme, limonoate:NAD(P) oxidoreductase from Pseudomonas sp. 321-18 cultured on a nutrient medium containing limonoate and use of the enzyme to debitter citrus products.
The primary disadvantage of the previously-isolated limonoid-metabolizing bacteria is that they all require the presence of a limonoid inducer in their growth media to produce cells which possess limonoid-metabolizing enzymes. Presently, no commercial source of citrus limonoids exists, thus production of bacterial cells for metabolizing limonoids is difficult, time-consuming and costly.